Spermatogonial stem cells of the mammalian testis are cells that keep growing limitlessly in the adult body to serve as a source of spermatogenesis via meiosis. Spermatogonial stem cells are dispensed to inherit genes to the next generation. Because spermatogonial stem cells are the only type of stem cells found in the adult body, they are useful for in vivo experiments, medical research, and biotechnology.
Brinster et al. succeeded in transplanting spermatogonial stem cells in vivo in 1994 (see “Brinster R L, Zimmermann J W, Spermatogenesis following male germ-cell transplantation, Proc Natl Acad Sci USA, 1994, Vol. 91, pp. 11298-11302”). In this method, when stem cells are transplanted into a seminiferous tubule, which constitutes the testis, they colonize and cause spermatogenesis of donor cell origin, whereby offspring can be produced. Thereby, the new possibility for manipulating germ line cells, in addition to ES cells, was pioneered, and a new field called developmental engineering using spermatogonial stem cells was established. Furthermore, a recent study reported that some spermatogonial stem cells are capable of surviving in vitro for 3 months or more (see “Nagano M, Avarbock M R, Leonida E B, Brinster C J, Brinster R L, Culture of mouse spermatogonial stem cells, Tissue Cell, 1998, Vol. 30, pp. 389-397”). In “Feng L-X, Chen Y, Dettin L, Reijo Pera R A, Herr J C, Goldberg E, Dym M, Generation and in vitro differentiation of a spermatogonial cell line, Science, 2002, Vol. 297, pp. 392-395”, “van Pelt A M M, Roepers-Gajadien H L, Gademan I S, Creemers L B, de Rooij D G, van Dissel-Emiliani F M F, Establishment of cell lines with rat spermatogonial stem cell characteristics, Endocrinology, 2002, Vol. 143, pp. 1845-1850” and Japanese Patent Kohyo Publication No. 2001-517927 (publication date: Oct. 9, 2001), methods of growing or long maintaining spermatogonial stem cells are described.
Meantime, methods of preparing transgenic animals (particularly knockout animals) using various biotechnological techniques have recently been developed, and are utilized for preparation of knockout animals, breed improvement of domestic animals, and the like. As examples of such methods of preparing a transgenic animal, the somatic cell nuclear transfer method, the method using ES cells, the method of gene injection to pronucleus, and the like can be mentioned.
Although the somatic cell nuclear transfer method is considered to be the only currently available method enabling production of knockout animals in domestic animals such as bovines and swine, it is problematic in that the efficiency is very low, malformations are prevalent, and it is hence expensive.
The method using ES cells is now effectively utilized for mice, since it enables convenient, efficient preparation of knockout animals. However, in any animals other than mice (for example, domestic animals such as swine and bovines, and primates), no ES cells capable of producing germ cells have been collected to date, nor is there any report of knockout achieved by this technique. Additionally, preparing germ line cells using ES cells is also problematic in that the ES cells are likely to differentiate into cells other than germ line and thus lose the capacity of germ cell formation.
Additionally, the method of gene injection to pronucleus is a standard method for preparation of transgenic mice, and is already in practical use. For animals other than mice, however, the success rate is very low (for example, around 1% for swine and 1% or less for bovines) and the method is very expensive and unrealistic.
Provided that the above-described spermatogonial stem cells can be utilized in such preparation of transgenic animals, the same technique as with ES cells would be applicable because stem cells are capable of growing limitlessly, leading to the expectation that knockout animals can be prepared conveniently and efficiently.
However, there have been no successful attempts to grow these cells in vitro to the extent that permits practical application thereof, and to manipulate them.
Specifically, with the method described in “Nagano M, Avarbock M R, Leonida E B, Brinster C J, Brinster R L, Culture of mouse spermatogonial stem cells, Tissue Cell, 1998, Vol. 30, pp. 389-397”, spermatogonial stem cells reportedly survived in vitro for 3 months or more, but no evidence for stem cell proliferation is given. Additionally, the methods of cultivation described in Japanese Patent Kohyo Publication No. 2001-517927, “Feng L-X, Chen Y, Dettin L, Reijo Pera R A, Herr J C, Goldberg E, Dym M, Generation and in vitro differentiation of a spermatogonial cell line, Science, 2002, Vol. 297, pp. 392-395”, “van Pelt A M M, Roepers-Gajadien H L, Gademan I S, Creemers L B, de Rooij D G, van Dissel-Emiliani F M F, Establishment of cell lines with rat spermatogonial stem cell characteristics, Endocrinology, 2002, Vol. 143, pp. 1845-1850” and the like are problematic in that extraneous genes cannot be stably introduced to spermatogonial stem cells, and are also problematic in that no offspring derived from the spermatogonial stem cells cannot be obtained. Although spermatogonial stem cells can survive when cultured in vitro using one of the above-described methods of cultivation, the number of cells decreases to about 20% of the original number in 1 week under the present situation, and it is impossible to grow the cells. Hence, the conventional techniques are subject to limitation in manipulating spermatogonial stem cells to apply for biotechnology and the like. Additionally, no cases of actual spermatogenesis using spermatogonial stem cells cultured in vitro persistently for a long time have been reported to date.